The development of the recombinant DNA techniques has permitted the use of several microorganisms as host for the expression of heterologous proteins with pharmaceutical and industrial application.
Many different host cells are used today for the production of such heterologous proteins. Successful production of recombinant proteins has been accomplished with eukaryotic hosts. The most prominent examples are budding yeasts like Saccharomyces cerevisiae, Pichia pastoris or Hansenula polymorpha, filamentous fungi like Aspergillus awamori or Trichoderma reesei, or mammalian cells like e.g. CHO cells.
Yeasts are attractive hosts for the production of recombinant proteins and small metabolites. Pichia pastoris, a methylotrophic yeast, is frequently used as an expression system for the production of recombinant proteins, and more recently also for the production of small metabolites (Marx et al. Microb Cell Fact 7:23 (2008)). Pichia has a high growth rate and is able to grow on a simple, inexpensive medium. Pichia can grow in either shake flasks or a fermenter, which makes it suitable for both small and large scale production. Pichia pastoris has recently been reclassified into a new genus, Komagataella, and been separated into three new species: Komagataella pastoris, K. phaffii, and K. pseudopastoris (Kurtzman C P. Int J Syst Evol Microbiol 55, 973-976. (2005)). Therefore, Pichia pastoris is a synonym for all three species, K. pastoris, K. phaffii and K. pseudopastoris. In accordance with previous literature, Pichia pastoris is used throughout this text, implicitly meaning any of the Komagataella species. Similarly, Hansenula polymorpha and Pichia angusta are synonyms.
In most cases, host cells are cultivated in fed batch processes for industrial production. The overall productivity of such a process is a function of the integral of biomass over time and the specific productivity (qP) of the biomass. qP correlates with the specific growth rate (μ), usually continuously increasing with increasing μ. Therefore, high qP is achieved at high μ, whereas the optimum biomass-time integral (A) is achieved with high initial and then very low μ. This is reflected by the following formula to calculate the product yield (P) at constant qP:P=A·qP 
FIG. 1 shows the relation of qP and μ in P. pastoris (Maurer et al. 2006, Micr. Cell Fact. 5:37 doi: 10.1186/1475-2859-5-37).
Hence optimum productivity is achieved with a compromise of μ, usually controlled in fed batch by limited substrate feed.
A typical case of fed batch process is the production of recombinant proteins with microorganisms or mammalian cells. While the description of product concentration in the cell mass is rather straight forward in the case of an intracellular product, it is more complex to predict the kinetics of a secreted product. A typical case for secretion systems are recombinant yeasts. As the production of many proteins in yeasts is quite cost sensitive, efforts are made to predict and control productivity, process time and product titers. Approaches to optimize fed batch processes for the methylotrophic yeast Pichia pastoris have been described (Zhang et al. Biotechnol. Prog. 2005, 21: 386-393, Maurer et al. 2006, Micr. Cell Fact.).
The variable costs of a bioprocess correlate with the volumetric capacity of the required fermentation unit, and the process time this unit is required to produce a defined amount of the product. Thus, the volumetric productivity QP is the most plausible target for optimization. At a given process time point t, QP is defined as:QP=P/(V·t)
The cell cycle, or cell-division cycle, is the series of events that takes place in a cell leading to its division and duplication (replication).
Eukaryotic cell division proceeds through a highly regulated cell cycle comprising consecutive phases termed G1 (gap 1), S (synthesis), G2 (gap 2) and M (mitosis).
The phase G0 is called resting phase, where resting cells will, under certain circumstances or after receiving specific stimuli, initiate the synthesis of RNA and proteins (G1-phase) which are necessary to effectively carry out the multiplication of its DNA and the division of the cell into two daughter cells. Subsequently, DNA synthesis begins (S-phase); once the cell has duplicated its DNA, a second late-protein-synthesis period begins (G2-phase), which is the short phase preparing the cell for division (M-phase). G2 and M phase are both characterized by the double chromosome set and are often regarded together as G2+M phase.
During the brief phase of mitosis the eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets in two daughter nuclei. Mitosis is generally followed immediately by cytokinesis, separating the cytoplasm into two daughter cells to provide for equal shares of the cellular components.
After cell division, each of the daughter cells begins the interphase of a new cycle. Cells that have stopped dividing, temporarily or not, are said to have entered a state of quiescence or senescence (G0).
Cell cycle progression is tightly regulated by defined temporal and spatial expression, localisation and destruction of a number of cell cycle regulators, which exhibit highly dynamic behaviour during the cell cycle. For example, at specific cell cycle stages some proteins translocate from the nucleus to the cytoplasm, or vice versa, and some are rapidly degraded. For details of known cell cycle control components and interactions, see Alberghina L, Coccetti P, Orlandi I. Systems biology of the cell cycle of Saccharomyces cerevisiae: From network mining to system-level properties. Biotechnol Adv. 2009 November-December; 27(6):960-78. The cell cycle process is complex and highly regulated. Errors in the cell cycle can either kill the cell through apoptosis or may lead to uncontrolled cell division, and in some cases to cancer.
Cell cycle analysis, mainly through the study of the distribution of cells throughout the G0/G1, S and G2/M cell cycle phases has proven to be of use in the analysis of tumor samples and the study of the proliferative response to different stimuli as well as in other areas.
The timing and inter-dependence of DNA replication (S-phase) and mitosis (M-phase) are controlled by oscillations in the activities of cyclin-dependent kinases (Cdks). Higher eukaryotes have multiple Cdks whereas in yeasts, cell cycle progression requires a single Cdk known as Cdc2 in fission yeast and Cdc28 in budding yeast. Waves of kinase activities are determined to a large extent by cell cycle-dependent synthesis and degradation of Cdk's regulatory cyclin subunits. Entry into M-phase depends on the appearance of B-type cyclins whose associated kinase activity promotes formation of the mitotic spindle. In budding yeast two pairs of related B-type cyclins appearing during S-phase (Clb3,4) and G2 (Clb1,2) are involved in formation and elongation of the spindle.
Cross et al. (Molecular Biology of the Cell (2005) 16:2129-2138) describe a quantitative behaviour of the eukaryotic cell cycle control system depending on the level of Clb2 expression. A loss of robustness of a Clb2 overexpressing system was predicted.
A series of fungal regulators, including cell cycle regulators, were described to improve the yield of fungal metabolite production (WO01/29073).
In an effort to improve protein expression from a producer cell line recombinant p21 or another cell cycle inhibitor protein has been co-expressed to enhance single cell productivity (WO02/099100A2). p21 is a universal inhibitor of cyclin kinases conferring stable and quantitative cell cycle arrest. Thus, care has to be taken to avoid triggering cell death or apoptosis in addition to its cytostatic effect.
WO0216590A2 discloses the extension of protein biosynthesis of a cell culture by switching the cells from a replicative to a productive state (RP switch), which is a pseudosenescent state. This can be accomplished by transformed cells conditionally expressing a cell cycle blocker arresting cell division. By preventing cell proliferation inducing differentiation to a senescence-like state, increased yields of bioproducts would be obtained.
Several methods can be used to synchronise cell cultures by halting the cell cycle at a particular phase, or separating cells of different phases. For example, serum starvation or addition of alpha factor would halt the cell in the G1 phase, mitotic shake-off, treatment with colchicine and treatment with nocodazole halt the cell in M phase and treatment with 5-fluorodeoxyuridine halts the cell in S phase.
A common measure to prolong the production phase of a cell culture is the limitation of substrates once biomass has grown to a certain extent. Likewise, additives to culture media are described to influence the cell cycle. KR100254810B1 discloses the addition of the antibiotic novobiocin to a CHO cell culture to increase the production of recombinant erythropoietin. Novobiocin serves as an inhibitor of early phases (pre-M) of the cell cycle.
Uchiyama et al. (Biotechnol Bioeng 54:262-271 (1997)) describe synchronous and arrested cultures of Saccharomyces cerevisiae. Synchrony was induced using both temperature-sensitive cdc mutants and inhibitors to arrest cell cycle progression to study cell cycle dependency. The cell cycle was stopped by switching the temperature from a permissive to a repressive one or else by the addition of a cell cycle inhibitor.
The universal blocking of cell growth and proliferation could effectively lead to a cell arrest and early apoptosis, resulting in a short production period of the cell culture. In general, the prolonged productivity in the absence of cell growth can hardly be maintained with state of the art technology.
It is the object of the present invention to prolong a highly productive phase of a cell culture to increase the yield of bioproducts.